Arabinogalactans are polysaccharides composed of arabinose and galactose, and exist in a variety of plants (such as Larix occidentalis, Baptisia tinctoria, Echinacea purpurea, and Curcuma longa). It has been reported that arabinogalactans are effective in treating intestinal disorders (such as diverticulosis and irritable bowel syndrome), enhancing the activity of the human immune system, and inhibiting metastasis of liver tumor cells, thereby being able to serve as dietary fiber, an immunomodulator, and a therapeutic agent for cancer (Pal, A. (2008). Chapter 13. Arabinogalactan Protein and Arabinogalactan: Biomolecules with Biotechnological and Therapeutic Potential. In K. G. Ramawat and J. M. Merillon (Eds.) Bioactive molecules and medicinal plants (pp 255-270) Springer).
Arabinogalactans are classified into the following two categories according to structure:    (1) type I arabinogalactans (also referred to as arabino-4-galactan), which contain 1,4-linked galactose residues serving as a backbone and forming a straight chain; and    (2) type II arabinogalactans (also referred to as arabino-3,6-galactan), which contain 1,3-linked or 1,6-linked galactose residues serving as a backbone, and which are normally attached to a protein to form an arabinogalactan protein.
Arabinogalactan proteins have been successfully isolated from organisms by the researchers in this field. For instance, as reported in Birgit Classen et al. (2000), Carbohydrate Research, 327:497-504, the pressed juice of Echinacea purpurea was subjected to tangential cross flow filtration to obtain a high molecular weight fraction, and the β-glucosyl Yariv reagent was subsequently used to perform precipitation on the high molecular weight fraction, such that an arabinogalactan protein was isolated. The arabinogalactan protein was subjected to molecular weight determination, chemical composition analysis, and linkage analysis. It was found from the results of molecular weight determination that the arabinogalactan protein has a molecular weight of 1.2×103 kDa. Furthermore, it was found from the results of chemical composition analysis that the arabinogalactan protein contains 83% (w/w) of a type II arabinogalactan, which has a molar ratio of galactose to arabinose being 1.8:1. In addition, it was found from the results of linkage analysis that most of the galactose residues of the arabinogalactan protein are 3,6-linked galactose residues.
As described in Kiyoshi Mashiguchi et al. (2004), Plant Cell Physiol., 45:1817-1829, rice bran of japonica rice was homogenized, and the resultant homogenate was subsequently centrifuged, followed by collecting the supernatant thus formed. Precipitation was conducted using ethanol, and 20 mM Tris-HCl (containing 1% (v/v) Triton X-100; pH 8) was added to the resultant precipitate, followed by centrifugation. The resultant supernatant was collected, and CaCl2 was added to the collected supernatant, followed by centrifugation. The supernatant thus formed was collected and was subjected to dialysis against deionized water. Afterward, a concentration process was conducted under vacuum. A 1 mM β-glucosyl Yariv reagent (β-GlcY) and 1% (w/v) NaCl were added to the resultant concentrate, followed by centrifugation. The pellet thus formed was collected, and dimethyl sulfoxide, sodium dithionate, and water were added thereto, such that a pale yellow solution was obtained. Subsequently, dialysis was conducted against deionized water. A solution containing β-glucosyl Yariv-reactive proteins was obtained and was subjected to reverse phase high performance liquid chromatography (RP HPLC). Consequently, three fractions, i.e. Fr. A to Fr. C, were obtained. Fr. A was subjected to deglycosylation, and the resultant deglycosylated Fr. A was subjected to N-terminal sequencing. The sequencing data thus obtained were compared with the known sequences in the rice cDNA database of KOME (knowledge-based Oryza molecular biological encyclopedia), thereby verifying that the β-glucosyl Yariv-reactive proteins in Fr. A are classical arabinogalactan proteins.
As disclosed in WO 2011/139168 A1, each of five honey samples (i.e. 0.5 year-old manuka honey, 2.5 year-old manuka honey, 5 year-old manuka honey, 1.5 year-old kanuka honey, and 1 year-old clover honey) was subjected to centrifugal ultrafiltration, followed by collecting fractions having a molecular weight greater than 10 kDa. Therefore, high molecular weight fractions were obtained. Subsequently, the high molecular weight fractions were subjected to salt precipitation, and the resultant precipitates were removed. A further precipitation process was conducted using the Yariv reagent, such that arabinogalactan proteins were obtained. It was proved by in vitro cell study that the arabinogalactan proteins stimulate the release of TNF-α from cells and hence have a pro-inflammatory effect. Furthermore, via gas chromatography-mass spectrometry analysis (GC-MS analysis) and glycosyl linkage analysis, it was proved that the high molecular weight fractions contain a type II arabinogalactan.
In addition, the researchers in this field have found that type II arabinogalactans exist in organisms. For instance, as reported in Esther Marie Goellner et al. (2011), Carbohydrate Polymers, 86:1739-1744, wood chips of Larix laricina were subjected to aqueous extraction, and an aqueous extract was hence obtained. Afterward, ethanol was added to the aqueous extract so as to form a precipitate having a high molecular weight, and the precipitate was subjected to molecular mass determination, monosaccharide composition analysis, and linkage analysis. It was verified by the results thus obtained that the precipitate contains a type II arabinogalactan. Particularly, it was found from the results of monosaccharide composition analysis that the type II arabinogalactan in the precipitate is composed of galactose, arabinose, and a small amount of glucuronic acid, and has a molar ratio of galactose to arabinose being 6:1. Moreover, it was found from the results of linkage analysis that the type II arabinogalactan in the precipitate contains 3,6-linked galactose residues, 1,6-linked galactose residues, terminally linked galactose residues, and a small amount of 1,3-linked galactose residues.
TW I379688 B1 discloses a polysaccharide extract of Anoectochilus formosanus for stimulating the release of granulocyte colony-stimulating factor (G-CSF). The polysaccharide extract of Anoectochilus formosanus is prepared using the following method. A sample of Anoectochilus formosanus is subjected to extraction with water so as to obtain a water-soluble extract. Subsequently, ethyl acetate is added to the water-soluble extract so as to conduct a partitioning process. The resultant aqueous phase is collected, and ethanol is added thereto. Afterward, the resultant precipitate is acquired and is dissolved in water. Furthermore, as shown in the examples of TW I379688 B1, the polysaccharide extract of Anoectochilus formosanus thus obtained was subjected to enzymatic hydrolysis using amylase, amyloglucosidase, and protease, followed by adding ethanol to the resultant hydrolysate. After the thus obtained precipitate was dissolved in water, a polysaccharide yet to be purified and having an average molecular weight of 29 kDa (hereinafter referred to as Anoectochilus formosanus polysaccharide) was acquired. It has been verified via experiments that Anoectochilus formosanus polysaccharide contains a type II arabinogalactan, and is capable of stimulating macrophages to release nitrogen monoxide and G-CSF, as well as reducing the concentration of tumor necrosis factor-α (TNF-α) in the blood of mice. Therefore, Anoectochilus formosanus polysaccharide is effective in activating innate immune cells and has anti-inflammatory activity.
Yang L. C. et al. (2013), Evid. Based Complement Alternat. Med., 2013:458075 and Yang L. C. et al. (2014), Phytomedicine, 21:647-655 are two relevant journal articles which describe the follow-up study conducted by the research team for the invention of TW I379688 B1. As reported in Yang L. C. et al. (2013), supra, cells of mouse colon carcinoma cell line CT26 were inoculated into mice to induce colon cancer, followed by intraperitoneally injecting 5-fluorouracil (5-FU) into the mice and orally administering Anoectochilus formosanus polysaccharide so as to investigate the synergistic anticancer effect of Anoectochilus formosanus polysaccharide and 5-FU on colon cancer cells. The experimental results indicate that Anoectochilus formosanus polysaccharide is able to effectively reduce the leukopenia caused by 5-FU. As set forth in Yang L. C. et al. (2014), supra, Anoectochilus formosanus polysaccharide was further purified by virtue of anion exchange chromatography, such that a type II arabinogalactan was obtained. It was proved by experiments that the type II arabinogalactan has innate immuno-modulatory activity and anti-colon cancer activity.
Rice hulls are the protecting covering of grains of rice, and are normally used as building material, insulation material, fertilizer, and fuel. Through research, the inventor has obtained a refined product from a rice hull. The refined product consists essentially of a type II arabinogalactan having a number average molecular weight ranging from 56 to 103 kDa. Particularly, the type II arabinogalactan in the refined product has a physical/chemical property (e.g. the ratio of galactose to arabinose and the backbone) different from that of the conventional type II arabinogalactans in other organisms, and is effective in enhancing the biological activity of innate immune cells and in treating allergy and caner (in particular colorectal cancer, breast cancer, lung cancer, and lymphoma).